Structural studies of T cell receptors (TCRs) bound to peptide-MHC ligands have provided much information on TCR recognition of foreign and self antigens. However, major gaps in our knowledge remain. Although peptides containing post-translational modifications (phosphorylation, glycosylation, cysteinylation) can be recognized by T cells, it is unknown how TCRs detect these modifications. Furthermore, the mechanisms whereby TCR engagement by peptide-MHC initiates T cell signaling remain unclear. A key to solving this problem is establishing the structural organization of the TCR- MHC-CD4 and TCR-CD3 complexes. We propose a new approach, directed evolution by yeast surface display (YSD), to acquire this information. Finally, the evolutionary origin of adaptive immunity in vertebrates is the subject of much conjecture. We will obtain insights into this evolution by defining the binding mode of antigen receptors expressed by T cell-like lymphocytes of jawless vertebrates (lamprey). Our objectives are: 1. Basis for TCR recognition of tumor-associated phosphopeptides. Among post-translationally modified epitopes, phosphopeptides are of special interest because dysregulated phosphorylation is a hallmark of malignant transformation. To understand how phosphorylation alters the antigenic identity of self-peptides, we will examine TCR recognition of HLA- DR1-restricted MART-1 phosphopeptides from melanoma tumors. 2. Structure of a TCR-MHC-CD4 complex. Although structures of TCR-MHC and MHC-CD4 binary complexes have been reported, no structure of a TCR-MHC-CD4 ternary complex has been determined. A major obstacle to crystallizing this complex is its intrinsic instability. We previously engineered a stable TCR-MHC complex and now propose to increase the affinity of the MHC-CD4 interaction by YSD. High-affinity CD4 or MHC mutants will be used to assemble a TCR-MHC-CD4 complex for structural analysis. 3. Structure of TCR-CD3 complexes. The TCR-CD3 receptor complex is composed of TCR noncovalently associated with CD3 subunits. Although the extracellular regions of TCR and CD3 interact, all attempts to crystallize TCR- CD3 complexes have been frustrated by the very low affinities of these interactions. We will use YSD to isolate high-affinity TCR and CD3 variants to assemble stable TCR-CD3 complexes. Structural information on these complexes will define the spatial organization of the TCR-CD3 receptor. 4. Basis for antigen recognition by T cell-like lymphocytes of lamprey. Adaptive immunity in jawless vertebrates is mediated by variable lymphocyte receptors (VLRs). Two types of VLRs (A and B) are expressed by mutually exclusive lymphocyte populations. VLRB lymphocytes resemble B cells of jawed vertebrates, whereas VLRA lymphocytes are remarkably similar to T cells. To compare antigen recognition by VLRA and TCR anticipatory receptors, we will determine structures of specific VLRA-antigen complexes. PUBLIC HEALTH RELEVANCE: While remarkable progress has been made in understanding the molecular basis for TCR recognition of microbial and self antigens, much less is known about the mechanisms by which TCR ligation initiates T cell signaling. That is, how does a T cell first know when TCR has bound peptide-MHC? The goal of this proposal is to obtain information on the structural organization of the TCR-MHC-CD4 and TCR- CD3 complexes, which together mediate T cell activation. In addition, we will explore TCR recognition of phosphopeptides, a new category of tumor antigens with potential as targets for cancer immunotherapy.